PROJECT SUMMARY The goals of this R01 proposal include the molecular and immunological characterization of vaccine adjuvant activity associated with processes mediated by the protein Stimulator of Interferon Genes (STING). Two vital insufficiencies are currently evident regarding adjuvanted human vaccines. First, very few adjuvants are approved for clinical use in the U.S. Second, the precise mechanistic bases of adjuvant-associated immune augmentation are poorly understood. Effective adjuvants trigger rapid, localized innate immune responses following their administration. Fundamentally, the innate signaling is initiated through engagement of pattern recognition receptors (PRRs) by ligands indicative or imitative of microbial infection. This, in turn, leads to expression of immunomodulatory and proinflammatory factors that ultimately direct adaptive immune responses capable of eliminating infected tissues. STING represents the PRR that senses cyclic dinucleotides (CDN), a product of the cellular enzyme cyclic GMP-AMP synthase (cGAS) following its detection of cytoplasmic dsDNA derived from microbes, mitochondria, or the nucleus. STING-mediated phenotypes are ultimately conferred by genes that are transcriptionally induced by the activated protein. This crucially involves the transcription factors IFN regulatory factor 3 (IRF3) and nuclear factor ?B (NF-?B), which synthesize mRNAs of distinct ontologies yet whose functional roles are mostly unexplored. Moreover, STING appears to control physiological processes that differ dramatically between cell types of the immune system including stromal, myeloid, and T cells. Intriguingly, pharmacologic induction of STING-dependent activity in murine models greatly enhances vaccine efficacy as indicated by protective immunity elicited against diverse pathogens. Unfortunately, the precise molecular and innate correlates of adaptive immune potentiation associated with STING activity remain largely unexamined. Furthermore, whether STING adjuvants elicit similarly effective immunogenic outcomes in primates has not been examined. We plan to couple these with our powerful CRISPR and transcriptomic technologies to obtain penetrative insight into the fundamental bases of STING-mediated immune outcomes. We hypothesize that the enhancement of antigen-directed adaptive immunity associated with STING-based adjuvants is functionally linked to molecular and cellular processes that are discernable using these models. We have also identified a first-in-class small molecule that activates cGAS-STING across species and enhances immunogenicity to Zika virus antigen. Including this alongside CDN in in vitro, murine, and nonhuman primate (NHP) models will allow us to: 1) Validate and characterize cGAS as a new immunotherapeutic target; 2) Demonstrate STING adjuvant efficacy in a highly clinically relevant model species; and 3) Identify species-specific similarities and differences with respect to STING-mediated immune responses.